Volume 3, Issue 1, Pages 11-21 (January 1999) Altered Trafficking of Lysosomal Proteins in Hermansky-Pudlak Syndrome Due to Mutations in the β3A Subunit of the AP-3 Adaptor  Esteban C Dell’Angelica, Vorasuk Shotelersuk, Ruben C Aguilar, William A Gahl, Juan S Bonifacino  Molecular Cell  Volume 3, Issue 1, Pages 11-21 (January 1999) DOI: 10.1016/S1097-2765(00)80170-7

Figure 1 Immunofluorescence Microscopy Analysis of AP-3 Expression in Fibroblasts from a Normal Individual and from Two HPS Patients Fibroblasts in primary culture from a normal individual (A, D, G, J, and M), a patient with mutations in HPS1 (patient 8) (B, E, H, K, and N), and patient 40 (C, F, I, L, and O) were fixed, permeabilized, and incubated with primary rabbit antibodies to the δ (A–C), β3 (D–F), or σ3 (G–I) subunits of AP-3, the γ subunit of AP-1 (J–L), or the α subunit of AP-2 (M–O). The primary antibodies were revealed by incubation with either Cy3-conjugated anti-rabbit immunoglobulins (A–I) or Alexa448-conjugated anti-mouse immunoglobulins (J–O) secondary antibodies. Images were obtained by confocal fluorescence microscopy using identical parameters for the three cell samples. Notice the punctate cytoplasmic staining for AP-3 subunits in normal (A, D, and G) and patient 8 (B, E, and H) cells, and the absence of this pattern in patient 40 cells (C, F, and I). Bar, 20 μm. Molecular Cell 1999 3, 11-21DOI: (10.1016/S1097-2765(00)80170-7)

Figure 2 Expression Levels of AP-3 Subunits in Fibroblasts from HPS Patient 40 as Determined by Immunoblot or Northern Blot Analysis (A) Fibroblast extracts from a normal individual (N), a patient with mutations in HPS1 (P8), or patient 40 (P40) were resolved by SDS-PAGE and analyzed by immunoblotting with antibodies to the δ, β3, μ3, or σ3 subunits of AP-3, the γ subunit of AP-1, the α subunit of AP-2, or the small GTP-binding protein ARF, as indicated in the figure. Notice the reduced levels of all four subunits of AP-3 and the normal levels of AP-1 γ, AP-2 α, and ARF in cells from patient 40. (B) Triton X-100 extracts of fibroblasts from a normal individual (N) and from patient 40 (P40) were fractionated on a Superose 6 gel-filtration column. The fractions were analyzed by immunoblotting for AP-3 β3 and AP-2 α. The positions of molecular size markers (Stokes’ radii given in Ångstroms) and the void volume (Vo) are indicated. Despite the markedly different levels, β3 peaked at fractions #32–34 in both patient 40 and normal cells. (C) Northern analyses showing normal expression of AP-3 subunit mRNAs in cells from patient 40. Total RNA extracted from fibroblasts from a normal individual (N), patient 8 (P8), or patient 40 (P40) were resolved on formaldehyde gels, transferred to nylon membranes, and probed with 32P-labeled DNA probes to β-actin (control), δ, β3A, μ3A, σ3A, or σ3B, as indicated. Molecular Cell 1999 3, 11-21DOI: (10.1016/S1097-2765(00)80170-7)

Figure 3 Mutations of the β3A Gene in HPS Patients (A) Schematic representation of the β3A subunit of AP-3 showing the amino-terminal (“A”), hinge (“H”), and carboxy-terminal (“C”) domains of the protein (Dell'Angelica et al. 1997b). The amino acid numbers demarcating the boundaries of the different regions are shown on top. The scheme also shows the approximate positions of a 21-amino acid deletion (Δ390–410) and a single amino acid substitution (L580R) found in patients 40 and 42. (B) Sequence of the Δ390–410 deletion allele. (C) Sequence of the L580R single amino acid substitution allele indicating the position of the AluI site that is abrogated by the point mutation. (D) Pedigree of the family of patients 40 and 42. (E) Agarose gel electrophoretic analysis of the deletion mutation. Notice the presence of a 486 bp band corresponding to the deletion allele in the RT-PCR products obtained from the father (lane 1) and the four siblings (lanes 3–6). (F) Agarose gel electrophoresis of a 288 bp RT-PCR product including the site of the point mutation. (G) Agarose gel electrophoretic analysis of the fragments shown in (F) after digestion with AluI. The 194 bp fragment corresponds to the point mutated allele (L580R), which was detected in the mother (lane 2) and patients 40 and 42 (lanes 3 and 4). Molecular Cell 1999 3, 11-21DOI: (10.1016/S1097-2765(00)80170-7)

Figure 4 Enhanced Degradation of β3A and σ3A in Cultured Fibroblasts from Patient 40 (A) Cultures of normal (N) and patient 40 (P40) fibroblasts were metabolically labeled for 20 min with [35S]methionine and chased for the times indicated in the figure, either in the absence or in the presence of the proteasome inhibitor LLnL (0.1 mM). Cells were detergent-solubilized under denaturing conditions and β3A and σ3A isolated by immunoprecipitation. Samples were analyzed by SDS-PAGE and fluorography, and bands were quantified by densitometric scanning. Each data point represents the mean of duplicate samples. (B) The biosynthesis of AP-3 β3A or AP-1 γ in normal (N) and patient 40 (P40) fibroblasts labeled for 20 min was measured as described in (A) and normalized to the total amount of [35S]methionine incorporated into proteins, as determined by trichloroacetic acid precipitation. The results are expressed as the mean ± SEM of the number of determinations shown in parentheses. (C) β3A was isolated by immunoprecipitation from normal (N) and patient 40 (P40) fibroblasts pulse-labeled for 20 min and chased for 0 or 1 hr as described in (A). Immunoprecipitates were incubated in the absence or presence of alkaline phosphatase prior to analysis by SDS-PAGE. Notice the alkaline-phosphatase-induced shift in electrophoretic mobility of β3A after the 1 hr chase in both normal (N) and patient 40 (P40) fibroblasts. Molecular Cell 1999 3, 11-21DOI: (10.1016/S1097-2765(00)80170-7)

Figure 5 Increased Surface Expression of CD63 in AP-3 Deficient Cells (A–B) Immunofluorescence microscopy analysis of the distribution of endogenous CD63 in normal (A) and patient 40 (B) fibroblasts permeabilized with saponin. (C–D) Immunofluorescence microscopy analysis of the expression of endogenous CD63 on the surface of nonpermeabilized normal (C) and patient 40 (D) fibroblasts. (E–F) Immunofluorescence microscopy imaging of normal (E) and patient 40 (F) fibroblasts allowed to internalize antibodies to CD63 for 15 min at 37°C. Bar, 20 μm. Molecular Cell 1999 3, 11-21DOI: (10.1016/S1097-2765(00)80170-7)

Figure 6 Differential Effects of AP-3 Deficiency on Surface Expression and Internalization of Lysosomal and Nonlysosomal Proteins (A) Analysis of the surface expression of CD63, lamp-1, lamp-2, TfR, and M6PR in normal (empty bars) or patient 40 (solid bars) fibroblasts as determined by immunofluorescence staining of nonpermeabilized cells followed by confocal microscopy and computer-assisted image analysis. Values of fluorescence intensity per cell (mean ± SD, three independent experiments) are relative to those of normal fibroblasts. Student’s t-test: *, p < 0.05. (B) Quantitation of antibody internalization experiments by computer-assisted measurement of the fluorescence per cell of samples prepared as in Figure 5E and Figure 5F. Values are relative to those obtained for the normal control and represent means ± S. D. of three independent experiments. Student’s t-test: *, p < 0.05. (C) Flow cytometric analysis of the expression of CD63, TfR, and MHC class I molecules on the surface of fibroblasts from a normal individual (thin, filled curve) and patient 40 (thick, unfilled curve). (D) Flow cytometric analysis of the expression of CD63 in B-lymphoblastoid cell lines from a normal individual, patients 40 and 42, and the patients’ father (heterozygous Δ390–410). Molecular Cell 1999 3, 11-21DOI: (10.1016/S1097-2765(00)80170-7)

Figure 7 Yeast Two-Hybrid Analysis of the Interaction of the Sorting Signals of CD63, lamp-1, and TfR with Different Adaptor Subunits GAL4 transcription activation domain fused to β2, μ1, μ2, or μ3A were coexpressed in yeast cells with GAL4 DNA-binding domain fused to a TGN38 tail-derived segment bearing either an inactive sequence (SDAQRL, control) or the tyrosine-based sorting signals of CD63 (SGYEVM), lamp-1 (AGYQTI), or TfR (LSYTRF). (A) Plate growth assay. Yeast cells were plated on minimal medium plates with (+His) or without (−His) histidine. Interactions were detected based on the ability of the cotransformed yeast cells to grow in the absence of histidine. (B) Growth inhibition assay. Interactions between the μ subunits and the signals of CD63, lamp-1, and TfR were further characterized by measuring growth of the cotransformed yeast cells after 2 days of culture in a minimal liquid medium lacking histidine and containing different concentrations of the histidine biosynthesis inhibitor 3-amino-1,2,4-triazole (3AT), as described (Aguilar et al. 1997). Molecular Cell 1999 3, 11-21DOI: (10.1016/S1097-2765(00)80170-7)